The first functional organ system that develops during the mammalian life is the circulatory system, which consists of the heart tube, great vessels and hematopoietic tissues. The heart tube serves as the pump that starts to beat even before the formation of effective circulatory loop, and the dorsal aortae serve as conduits that form independently from the heart tube and link to it soon after. These two organs are structurally and molecularly similar in that they consist of outer muscular and inner endothelial/endocardial layers, and that each layer expresses many of the genes in common. Therefore, although derived from developmentally distinct origins, the heart tube and the dorsal aorta likely share similar genetic program during their formation. The third component of the circulatory system, the hematopoietic cells, arises from endothelial cells in dorsal aorta. This process is closely associated with the arterial identity of the aortic endothelium, such as the expression of arterial markers and the influence of biomechanical forces generated by the heartbeat. Interestingly, the endocardium in the heart tube shares all these molecular and physiological properties with aortic endothelilum. It has been suggested that the early cardiac progenitors express hematopoietic genes, and that cardiac and hematopoietic lineages are closely related in non-mammalian and embryonic stem cell models. In this proposal, we will examine the developmental role of the heart as a hematopoietic site. We specifically propose to characterize the spatiotemporal distribution and the multi-lineage differentiation potential of the putative hemogenic cells in the forming endocardium (Aim 1) and to examine the role of Nkx2.5 during the endocardial development (Aim 2). The overall goal of this proposal is to test the hemogenic activity of the heart. Discovery of a new role of the heart and a hidden source of blood cells will significantly enhance our understanding of the developmental program of cardio-vasculo-hematopoietic development. Clinically, extra-medullary hematopoiesis is often observed in patients with heart diseases and hematological disorders. The project will shed a light on the pathogenesis of this clinicopathological disease entity. PUBLIC HEALTH RELEVANCE: The close relationship between cardiac and hematopoietic lineages has been suggested in developmental and disease contexts, but it is still unclear the biological significance of the hematopoietic program in the heart. Here, we will uncover the novel role of the heart as a hemogenic organ.